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CONSTRUCTION OF PRODUCTION SYSTEMS OF USEFUL COMPOUNDS IN PLANT CELL CULTURE BY APPLICATION OF PLANT GENE EXPRESSION SYSTEM

应用植物基因表达系统构建植物细胞培养有用化合物生产体系

基本信息

批准号：
04454037
负责人：
SHINMYO Atsuhiko
金额：
$ 1.66万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY (1994)Osaka University (1992-1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

We studied construction of a production system of useful compounds in plant cell culture by application of plant gene expression mechanism. Tobacco cells, Nicotiana tabacum L.cv. BY2, was selecteed as a hosat cell, since it grows quite faster among plant cells (doubling time is 10-11h). A set of target genes introduced into tobacco cells must be placed under the control of strong promoters, enhancers, and cis-elements respondiog to operation conditions of plant cell culture. A promoter of horseradish peroxidase isozyme gene, prxC2, showed high activity in tobacco cells. Expression of the prxC2 gene was induced by wounding, and a cis-element, of which core sequence was CACGTG,for wound-induction was identified at-289 bp from the translation point. A cDNA encodig a transcription factor associated with the cis-element was also cloned. The wound-induction system seems to be effective on gene expression in the tobacco cultured cells.A heat shock promoter of HSP18.2 gene of Arabidopsis thaliana was ligated to the structural gene for beta-glucuronidase (GUS), and introduced into chromosome of the tobacco cultured cells. Expression of the GUS gene was strongly induced when incubation temperature was shifted from 25ﾟC to37ﾟC.GUS mRNA was detected at 15 min after temperature ahift and was maximum at 2 h. Maximun GUS sctivity was found at 4 h. This result indicated that the HSP18.2 promoter is a good candidate for expression of foreign genes by the control of incubation temperature of tobacco cultured cells.Growth rate of eukaryotic cells is controlled by cell cycle regulating proteins, such as protein kinases, cyclins, and protein phosphatases. Analysis of these gene expression will be important to understand mechanism of cell growth and enhancement of growth rate of plant cells. In the present study, three cyclin cDNAs were cloned grom tobacco cells, and it was found that these cyclin genes belonged to G2 cyclins.

本研究应用植物基因表达机制，构建了植物细胞培养生产有用化合物的系统。烟草细胞，烟草（Nicotiana tabacum L.cv.）BY_2是一种生长速度较快的植物细胞，倍增时间为10- 11小时。将一组目的基因导入烟草细胞中，必须根据植物细胞培养的操作条件，将其置于强启动子、增强子和顺式元件的控制之下。辣根过氧化物酶同工酶基因启动子prxC 2在烟草细胞中表现出较高的活性。prxC 2基因的表达被创伤诱导，并且在距离翻译点-289 bp处鉴定了用于创伤诱导的顺式元件，其核心序列为CACGTG。还克隆了编码与顺式元件相关的转录因子的cDNA。将拟南芥HSP 18. 2基因的热激启动子与GUS结构基因连接，并导入烟草培养细胞染色体中。当温度从25 ℃升高到37 ℃时，GUS基因的表达被强烈诱导，温度升高后15 min检测到GUS mRNA，2 h时达到最大值。GUS活性在4 h时最高。这一结果表明，HSP 18.2启动子是一个很好的候选启动子，可以通过控制烟草培养细胞的温育温度来表达外源基因，真核细胞的生长速率受细胞周期调控蛋白如蛋白激酶、细胞周期蛋白和蛋白磷酸酶的调控。分析这些基因的表达对了解细胞生长机制和提高植物细胞生长速率具有重要意义。本研究从烟草细胞中克隆了3个细胞周期蛋白基因，发现它们均属于G2期细胞周期蛋白。